Analysis of Brain Phosphoproteome Using Titanium Dioxide Enrichment and High-Resolution LC-MS/MS

  • Jeffrey M. Sifford
  • Haiyan Tan
  • Hong Wang
  • Junmin Peng
Protocol
Part of the Neuromethods book series (NM, volume 127)

Abstract

This chapter outlines the process of performing phosphoproteomic studies of brain tissue: brain dissection, protein extraction and digestion, phosphopeptide enrichment, and peptide identification and quantification by LC-MS/MS. We describe a refined method for rapid, simple, and efficient TiO2-based phosphopeptide enrichment that relies on specific binding of the peptidyl phosphate group and TiO2, with free phosphate competitor added to reduce nonspecific binding. Integration of such a robust phosphopeptide enrichment method, powerful high-resolution LC-MS/MS, and multiplex isobaric labeling enables deep profiling of phosphoproteome with high sensitivity from biological samples, such as the human brain.

Key words

Mass spectrometry LC-MS/MS Proteomics Proteome Phosphoproteome Brain phosphopeptide enrichment Titanium dioxide Isobaric labeling 

References

  1. 1.
    Aebersold R, Mann M (2016) Mass-spectrometric exploration of proteome structure and function. Nature 537:347–355CrossRefPubMedGoogle Scholar
  2. 2.
    Zhang Y, Fonslow BR, Shan B, Baek MC, Yates JR 3rd (2013) Protein analysis by shotgun/bottom-up proteomics. Chem Rev 113:2343–2394CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Macek B, Mann M, Olsen JV (2009) Global and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev Pharmacol Toxicol 49:199–221CrossRefPubMedGoogle Scholar
  4. 4.
    Xia Q, Cheng D, Duong DM, Gearing M, Lah JJ, Levey AI, Peng J (2008) Phosphoproteomic analysis of human brain by calcium phosphate precipitation and mass spectrometry. J Proteome Res 7:2845–2851CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Xu P, Duong DM, Peng J (2009) Systematical optimization of reverse-phase chromatography for shotgun proteomics. J Proteome Res 8:3944–3950CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Edbauer D, Cheng D, Batterton MN, Wang CF, Duong DM, Yaffe MB, Peng J, Sheng M (2009) Identification and characterization of neuronal MAP kinase substrates using a specific phosphomotif antibody. Mol Cell Proteomics 8:681–695CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Tan H, Wu Z, Wang H, Bai B, Li Y, Wang X, Zhai B, Beach TG, Peng J (2015) Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome. Proteomics 15:500–507CrossRefPubMedGoogle Scholar
  8. 8.
    Ficarro SB, McCleland ML, Stukenberg PT, Burke DJ, Ross MM, Shabanowitz J, Hunt DF, White FM (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat Biotechnol 20:301–305CrossRefPubMedGoogle Scholar
  9. 9.
    Nuhse TS, Stensballe A, Jensen ON, Peck SC (2003) Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry. Mol Cell Proteomics 2:1234–1243CrossRefPubMedGoogle Scholar
  10. 10.
    Pinkse MW, Uitto PM, Hilhorst MJ, Ooms B, Heck AJ (2004) Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem 76:3935–3943CrossRefPubMedGoogle Scholar
  11. 11.
    Iliuk A, Martinez JS, Hall MC, Tao WA (2011) Phosphorylation assay based on multifunctionalized soluble nanopolymer. Anal Chem 83:2767–2774CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Ballif BA, Villen J, Beausoleil SA, Schwartz D, Gygi SP (2004) Phosphoproteomic analysis of the developing mouse brain. Mol Cell Proteomics 3:1093–1101CrossRefPubMedGoogle Scholar
  13. 13.
    Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villen J, Li J, Cohn MA, Cantley LC, Gygi SP (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A 101:12130–12135CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Motoyama A, Xu T, Ruse CI, Wohlschlegel JA, Yates JR 3rd (2007) Anion and cation mixed-bed ion exchange for enhanced multidimensional separations of peptides and phosphopeptides. Anal Chem 79:3623–3634CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Steen H, Kuster B, Fernandez M, Pandey A, Mann M (2002) Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway. J Biol Chem 277:1031–1039CrossRefPubMedGoogle Scholar
  16. 16.
    Pandey A, Podtelejnikov AV, Blagoev B, Bustelo XR, Mann M, Lodish HF (2000) Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc Natl Acad Sci U S A 97:179–184CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD, Comb MJ (2005) Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 23:94–101CrossRefPubMedGoogle Scholar
  18. 18.
    Larsen MR, Thingholm TE, Jensen ON, Roepstorff P, Jorgensen TJ (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4:873–886CrossRefPubMedGoogle Scholar
  19. 19.
    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127:635–648CrossRefPubMedGoogle Scholar
  20. 20.
    Kettenbach AN, Gerber SA (2011) Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments. Anal Chem 83:7635–7644CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Wang H, Yang Y, Li Y, Bai B, Wang X, Tan H, Liu T, Beach TG, Peng J, Wu Z (2015) Systematic optimization of long gradient chromatography mass spectrometry for deep analysis of brain proteome. J Proteome Res 14:829–838CrossRefPubMedGoogle Scholar
  22. 22.
    Eng JK, McCormack AL, Yates JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5:976–989CrossRefPubMedGoogle Scholar
  23. 23.
    Beausoleil SA, Villen J, Gerber SA, Rush J, Gygi SP (2006) A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol 24:1285–1292CrossRefPubMedGoogle Scholar
  24. 24.
    Wang X, Li Y, Wu Z, Wang H, Tan H, Peng J (2014) JUMP: a tag-based database search tool for peptide identification with high sensitivity and accuracy. Mol Cell Proteomics 13:3663–3673CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Taus T, Kocher T, Pichler P, Paschke C, Schmidt A, Henrich C, Mechtler K (2011) Universal and confident phosphorylation site localization using phosphoRS. J Proteome Res 10:5354–5362CrossRefPubMedGoogle Scholar
  26. 26.
    Mertins P, Yang F, Liu T, Mani DR, Petyuk VA, Gillette MA, Clauser KR, Qiao JW, Gritsenko MA, Moore RJ, Levine DA, Townsend R, Erdmann-Gilmore P, Snider JE, Davies SR, Ruggles KV, Fenyo D, Kitchens RT, Li S, Olvera N, Dao F, Rodriguez H, Chan DW, Liebler D, White F, Rodland KD, Mills GB, Smith RD, Paulovich AG, Ellis M, Carr SA (2014) Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels. Mol Cell Proteomics 13:1690–1704CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Oka T, Tagawa K, Ito H, Okazawa H (2011) Dynamic changes of the phosphoproteome in postmortem mouse brains. PLoS One 6:e21405CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Futterer CD, Maurer MH, Schmitt A, Feldmann RE Jr, Kuschinsky W, Waschke KF (2004) Alterations in rat brain proteins after desflurane anesthesia. Anesthesiology 100:302–308CrossRefPubMedGoogle Scholar
  29. 29.
    Ericsson C, Nister M (2011) Protein extraction from solid tissue. Methods Mol Biol 675:307–312CrossRefPubMedGoogle Scholar
  30. 30.
    Glatter T, Ludwig C, Ahrne E, Aebersold R, Heck AJ, Schmidt A (2012) Large-scale quantitative assessment of different in-solution protein digestion protocols reveals superior cleavage efficiency of tandem Lys-C/trypsin proteolysis over trypsin digestion. J Proteome Res 11:5145–5156CrossRefPubMedGoogle Scholar
  31. 31.
    Chen EI, Cociorva D, Norris JL, Yates JR 3rd (2007) Optimization of mass spectrometry-compatible surfactants for shotgun proteomics. J Proteome Res 6:2529–2538CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Pirmoradian M, Budamgunta H, Chingin K, Zhang B, Astorga-Wells J, Zubarev RA (2013) Rapid and deep human proteome analysis by single-dimension shotgun proteomics. Mol Cell Proteomics 12:3330–3338CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Li QR, Ning ZB, Tang JS, Nie S, Zeng R (2009) Effect of peptide-to-TiO2 beads ratio on phosphopeptide enrichment selectivity. J Proteome Res 8:5375–5381CrossRefPubMedGoogle Scholar
  34. 34.
    Liu H, Sadygov RG, Yates JR 3rd (2004) A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem 76:4193–4201CrossRefPubMedGoogle Scholar
  35. 35.
    Wang W, Zhou H, Lin H, Roy S, Shaler TA, Hill LR, Norton S, Kumar P, Anderle M, Becker CH (2003) Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards. Anal Chem 75:4818–4826CrossRefPubMedGoogle Scholar
  36. 36.
    Zhou JY, Afjehi-Sadat L, Asress S, Duong DM, Cudkowicz M, Glass JD, Peng J (2010) Galectin-3 is a candidate biomarker for amyotrophic lateral sclerosis: discovery by a proteomics approach. J Proteome Res 9:5133–5141CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386CrossRefPubMedGoogle Scholar
  38. 38.
    Hebert AS, Merrill AE, Bailey DJ, Still AJ, Westphall MS, Strieter ER, Pagliarini DJ, Coon JJ (2013) Neutron-encoded mass signatures for multiplexed proteome quantification. Nat Methods 10:332–334CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Werner T, Sweetman G, Savitski MF, Mathieson T, Bantscheff M, Savitski MM (2014) Ion coalescence of neutron encoded TMT 10-plex reporter ions. Anal Chem 86:3594–3601CrossRefPubMedGoogle Scholar
  40. 40.
    Thompson A, Schafer J, Kuhn K, Kienle S, Schwarz J, Schmidt G, Neumann T, Hamon C (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895–1904CrossRefPubMedGoogle Scholar
  41. 41.
    Ting L, Rad R, Gygi SP, Haas W (2011) MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods 8:937–940CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    McAlister GC, Nusinow DP, Jedrychowski MP, Wuhr M, Huttlin EL, Erickson BK, Rad R, Haas W, Gygi SP (2014) MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal Chem 86:7150–7158CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Savitski MM, Mathieson T, Zinn N, Sweetman G, Doce C, Becher I, Pachl F, Kuster B, Bantscheff M (2013) Measuring and managing ratio compression for accurate iTRAQ/TMT quantification. J Proteome Res 12:3586–3598CrossRefPubMedGoogle Scholar
  44. 44.
    Thompson AJ, Hart SR, Franz C, Barnouin K, Ridley A, Cramer R (2003) Characterization of protein phosphorylation by mass spectrometry using immobilized metal ion affinity chromatography with on-resin beta-elimination and Michael addition. Anal Chem 75:3232–3243CrossRefPubMedGoogle Scholar
  45. 45.
    Peng J, Elias JE, Thoreen CC, Licklider LJ, Gygi SP (2003) Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J Proteome Res 2:43–50CrossRefPubMedGoogle Scholar
  46. 46.
    Elias JE, Gygi SP (2007) Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4:207–214CrossRefPubMedGoogle Scholar
  47. 47.
    Bai B, Hales CM, Chen PC, Gozal Y, Dammer EB, Fritz JJ, Wang X, Xia Q, Duong DM, Street C, Cantero G, Cheng D, Jones DR, Wu Z, Li Y, Diner I, Heilman CJ, Rees HD, Wu H, Lin L, Szulwach KE, Gearing M, Mufson EJ, Bennett DA, Montine TJ, Seyfried NT, Wingo TS, Sun YE, Jin P, Hanfelt J, Willcock DM, Levey A, Lah JJ, Peng J (2013) U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer’s disease. Proc Natl Acad Sci U S A 110:16562–16567CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Rauniyar N, Yates JR 3rd (2014) Isobaric labeling-based relative quantification in shotgun proteomics. J Proteome Res 13:5293–5309CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Jeffrey M. Sifford
    • 1
    • 2
  • Haiyan Tan
    • 3
  • Hong Wang
    • 1
    • 2
  • Junmin Peng
    • 1
    • 2
    • 3
  1. 1.Department of Structural BiologySt. Jude Children’s Research HospitalMemphisUSA
  2. 2.Department of Neurodevelopmental BiologySt. Jude Children’s Research HospitalMemphisUSA
  3. 3.St. Jude Proteomics FacilitySt. Jude Children’s Research HospitalMemphisUSA

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